Hepatitis A virus (HAV) is responsible for over 20,000 cases of hepatitis in the United States each year. Certain populations are at high risk for infection with HAV including foreign travelers, children attending day care centers and their close contacts, military personnel, and persons with close contact of patients with hepatitis A. Currently, passive immunization with immune serum globulin (ISG) is the only effective measure for preventing HAV infections in these patients. ISG, however, elicits only low levels of neutralizing antibody and requires repeated doses.
An effective vaccine would be useful for active immunization of populations at high risk. Inactivated HAV vaccines have been developed which are immunogenic and protective against challenge with the live virus [see Binn et al., J. Infect. Dis., 153: 749-756 (1986) and Provost et al., Proc. Soc. Exp. Biol. Med., 159: 201-203 (1978)]. These vaccines may prove effective; however, they are usually costly, may fail to produce local (or secretory) immunity, and may require repeated administration. Live HAV vaccines are under development--Provost et al. have shown that a cell culture adapted variant of HAV, strain CR-326, is attenuated (infectious, but no longer causes disease) in nonhuman primates and induces protection against challenge with wild-type (naturally occurring) virus. The experiments which constitute the parent applications of this invention disclose the development of a cell culture adapted variant of HAV, strain HM-175, which is attenuated and which elicits serum neutralizing antibody providing protection against challenge with wild-type virus in chimpanzees.
Wild-type HAV, strain HM-175, grows poorly in cell culture; however, after several passages the virus adapts to cell culture and becomes attenuated for chimpanzees [see Feinstone et al, Develop. Biol. Standard, 54: 429-432 (1983)]. The biological basis for cell culture adaptation and attenuation of HAV is unknown. The complete nucleotide sequence of the genome of wild-type (naturally occurring) HAV, strain HM-175, has been disclosed in Cohen et al., "Complete nucleotide sequence of wild-type hepatitis A virus: comparison with different strains of hepatitis A virus and other picornaviruses." Comparison of the nucleotide sequence between wild-type HAV HM-175 and attenuated HM-175/7 indicates that a maximum of 25 nucleotide changes (resulting in 12 amino acid changes) are responsible for cell culture adaptation and attenuation of HAV-175.
While the biological basis for attenuation is unknown, three areas of the HAV genome may contain markers of attenuation. The RNAs of the 5' non-coding regions of wild-type HAV and HM-175/7 have different predicted secondary structures. The 5' non-coding region of HAV HM-175 Pass 35 has five nucleotide deletions (one 4-base and one single-base deletion) when compared to wild-type HM-175. The 5' non-coding region has the highest degree of nucleotide conservation among the different strains of HAV. However, when wild-type HM-175 and HM-175/7 are compared, 28% of the nucleotide differences (7/25 bases) are present in the 5' noncoding region, a region which contains less than 10% of the viral genome. Thus, this relatively small area of the genome may be important for both cell culture adaptation and attenuation.
The capsid region of the HAV genome may also be important for attenuation. HAV HM-175 Pass 35 has two amino acid differences from wild-type HM-175 in the capsid region. The nucleotide difference at position 3025 changes amino acid 273 of VP1 from glutamic acid (negatively charged) in wild-type HM-175 to valine (non-polar) in HM-175/7. Thus, one of the amino acid differences between wild-type and attenuated HAV HM-175 may occur at a site important for antibody binding.
The nucleotide sequences of other strains of HAV have been disclosed. A partial sequence of a variant of HAV HM-175 propagated in a cell culture not suitable for vaccine use is disclosed in Ross et al., "Molecular cloning of cDNA from hepatitis A virus strain HM-175 after multiple passages in vivo and in vitro," J. Gen. Virol. This strain was derived from wild-type virus after multiple passages (6 in vivo and 59 in vitro) and virus released into cell culture medium was used for molecular cloning. A different cell culture adapted strain of HAV (not known to be attenuated and not acceptable for vaccine production) is disclosed in Najarian et al., PNAS USA, 82: 627-631 (1985). Like HAV HM-175 Pass 35, the Ross et al variant contains a deletion of a single thymine at the nucleotide position 203-207. However, the Ross et al variant contains seven other nucleotide differences from wild-type HAV HM-175 in the 5' coding region which the variant of the present invention does not. The Najarian et al. strain has numerous differences from wild-type HAV HM-175 including an adenine at position 203 and a thymine at the 3' end of the genome. This strain, like the strain of the present invention, also contains a nucleotide difference at position 6522, resulting in an amino acid change from wild-type HAV HM-175 of a serine to a threonine. All of the cell culture adapted HAV stains that have been sequenced in the capsid region have nucleotide differences from wild-type HAV HM-175 at positions 1742 and 2864 that do not result in amino acid changes from the wild-type.